Method for preparing a separation medium

ABSTRACT

The present invention relates to a method of preparing a separation medium starting with an aqueous solution of spherical and preferably functionalised primary particles of defined size, comprising the following steps: a) inverse suspension dispersing said aqueous solution of primary particles as droplets in oil; b) evaporation to remove said aqueous solution and fusion between particles to form spherical aggregates; c) size fractionation of aggregates from step b); and optionally d) repeating steps a) to c) an optional number of times to form progressively larger spherical aggregates. The invention also relates to a separation medium produced by this method. The present separation medium can be used in chromatography in the form of essentially spherical aggregates in packed or expanded bed columns or in the form of assembled aggregates on a support for filtration purposes.

TECHNICAL FIELD

The present invention relates to a method of preparing a separationmedium, which is useful e.g. in chromatography and filtration. Further,the invention encompasses a novel separation medium as such and usethereof to separate a compound from a liquid. More closely, theinvention relates to fractal particles for chromatography media.

BACKGROUND OF INVENTION

Separation media, such as chromatography media and filtration media, areoften associated with non-satisfactory properties to some end. Importantfactors in this field are e.g. the mass transport properties of themedia, the flow properties thereof when used in chromatographic columnsor as membranes, cumbersome and non-reliable methods of preparation etc.Hence, there is an ongoing development to seek improvements in thisfield.

Reeder, et al. (An approach to hierarchically structured porous zirconiaaggregates. Reeder et al., Dep. Chem. Eng., Univ. Minnesota,Minneapolis, Minn., USA. Journal of Colloid and Interface Science(1996), 184(1), 328-330) disclose an approach to aggregate colloids intohierarchically structured spherical particles. Successive aggregationsteps are used to assemble an inorganic particle that is self-similar on2 size scales and is permeated by an ordered pore network with abidisperse size distribution. The structure of the micro- and macro-porenetworks as well as the mechanical integrity of the structure can becontrolled by varying sintering conditions. One drawback of thedisclosed aggregation is that the successive steps required will renderthe preparation thereof time-consuming and costly. This approach is alsolimited to inorganic particles that can be sintered.

U.S. Pat. No. 4,070,286 disclose a powder of discrete, macroporous,microspheroids each composed of a plurality of large colloidal particlesjoined and cemented together at their points of contact by non-porousamorphous silica. The cementing is done with a partial sinteringprocess, which is only possible for inorganic materials. Also, thecoacervation process described makes it difficult to control thesphericity of the aggregates.

O. D. Velev, K. Furusawa, K. Nagayama, have in Langmuir, 12, 2374-2384(1996), Langmuir, 12, 2385-2391 (1996) and Langmuir, 13, 1856-1859(1997) described assembly of latex particles into spherical aggregatesby using emulsion droplets as colloid templates. The aqueous latexsolution is dispersed by a homogenizer inside anhydrous octanol medium.The removal of water occurs by partitioning into the octanol phase. Thismeans that it starts as soon as the phases are brought into contact witheach other, leaving no time for controlled emulsification to produce therequired droplet size. The amount of water that can be removed is alsolimited by the equilibrium water solubility in the amount of octanolpresent.

U.S. patent application Ser. No. 09/341,181 relates to a cellulosicparticle body comprising interconnected cellulosic small particles withsmall interparticle spaces and to method for production thereofcomprising dispersing small cellulosic particles in an alkaline mediumand contacting the resulting suspension with a coagulating solution. Theprecipitation of the binder in the coagulating solution proceeds in anuncontrolled fashion and it is difficult to obtain desired open porestructures. The spraying processes involved also make it difficult toobtain aggregate sizes suitable for chromatography and in sufficientyield.

U.S. Pat. No. 3,782,075 discloses a packing material for chromatographycolumns, prepared from a powder of uniform-sized porous microspherescomposed of a plurality of interconnected colloidal oxide particles. Thesize of the pores is controlled by the size of the colloidal particlesused to form the microspheres and the surface area of the microsphere iscontrolled by the amount of sintering used to impart strength to theparticles. The primary particles are joined together by sintering, whichlimits the applicability to inorganic particles.

EP 0 442 977 relates to a chromatography method using a matrixcomprising interconnected first and second throughpore sets. The membersof the first throughpore set have a greater mean diameter than themembers of the second throughpore set. The second throughpore set is influid communication with solute interactive regions which interactreversibly with solutes to effect chromatographic separation thereof.The method of preparing the matrix appears unclear and not fullyreproducible which means that not all batches will show effects of ahierarchical structure. Furthermore, it may be difficult to controlsizes and connect throughpores with this method.

In spite of all prior art within this technical field, there is still aneed in this field of improved methods to prepare separation media aswell as of novel separation media.

Definitions

The term “hierarchical” or “fractal” is used herein to describe a porousstructure with large pores open to the exterior. On the walls of theselarge pores, a system of smaller pores opens. Optionally, a furthersystem of even smaller pores may open on the walls of these pores etc.

The term “separation medium” is used herein in a broad sense to includeany material that is useful as the stationary phase in a separationmethod, such as a chromatographic process or a filtration. The mediumcan be used as such or combined with another material, such as a rigidsupport in a filtration. Further, a “separation medium” as used hereinwill include both materials that are directly useful for adsorption orsieving and such materials having additional adsorbing groups, known asligands, coupled thereon.

SUMMARY OF INVENTION

One object of the invention is to provide a method of preparing aseparation medium of synthetic polymers with fractal or hierarchicalpore structure. The objective method should be fully controllable andthe pores fully accessible and permeable.

Another object of the invention is to provide a separation medium withimproved mass transport properties as compared to the prior art media.

A further object of the invention is to provide a separation medium,which is useful in chromatography, either in particle form, e.g. inpacked or expanded bed adsorption, or in the form of a membrane.

Yet another object of the invention is to provide a separation medium,which exhibits one or more ligands which may be of different kind incase of more than one type of ligand.

A further object of the invention is to provide a separation medium,which is especially suitable for use in expanded bed adsorption (EBA).This can be achieved according to the invention by a separation medium,the weight of which is suitable for expanded beds.

Thus, in a first aspect the invention relates to a method of preparing aseparation medium starting with an aqueous solution of essentiallyspherical primary particles of defined size, comprising the followingsteps:

-   -   a) inverse suspension dispersing said aqueous solution of        primary particles as droplets in oil;    -   b) evaporation to remove said aqueous solution and fusion        between primary particles to form spherical aggregates (beads);    -   c) size fractionation of aggregates from step b); and optionally    -   d) repeating steps a) to c) an optional number of times to form        progressively larger spherical aggregates. In case of repeating        steps a)-c), the aggregates from step c) are first suspended in        aqueous solution.

The primary particles are preferably 50-1000 nm in diameter.

The primary particles are made of a synthetic polymer selected from thegroup that consists of polymerised styrene and/or divinylbenzene,(meth)acrylates, vinyl esters, vinyl ethers, vinyl amides,meth(acrylamides), dienes, etc. or a mixture of two or more thereof.Preferably the primary particles are surrounded by a soft shell made offor example polyacrylate.

Preferably, the primary particles are functionalised with, for example,carboxy, epoxy or amino groups.

The fusion in step b) is preferably caused by a water-soluble polymerand a cross-linker in sufficient quantity, i.e. the minimum amount thatgives the required mechanical stability, such as at least a mass ratioof 0.3:100.

Preferably, the water-soluble polymer is polyethyleneimine and thecross-linker is chosen from N,N′-methylenebisacrylamide and diacryloylpiperazine.

Alternatively, the soft shells adhere to each other without any fusionpromoting agent. Yet another alternative, is thermal fusion of theparticles in step b).

In a second aspect, the invention relates to a separation mediumcomprised of one or more aggregates, wherein each aggregate comprises aplurality of porous and essentially spherical synthetic polymer beadsassembled into essentially spherical aggregates of controlled size.

The medium has a mass ratio of synthetic polymer:beads in each aggregateof about 0.1:100 to about 10:100, preferably from 0.3:100 to 3:100.

The beads comprise a synthetic polymer selected from the group thatconsists of polymerised styrene and/or divinylbenzene, (meth)acrylates,vinyl esters, vinyl ethers, vinyl amides, meth(acrylamides), dienes,etc. or a mixture of two or more thereof.

Preferably, the polymers have been chemically crosslinked or can becrosslinked at a later stage. Alternatively, the polymers may bethermally fused. The medium may exhibits one kind or two different kindsof ligand coupled to the beads within the aggregates.

An aggregate is comprised of at least about 10, and preferably at leastabout 20, beads and may contain up to 400 beads.

In the medium, the aggregates are separate and essentially sphericalentities, which medium is suitable for use in packed or expanded bedadsorption. The medium is also suitable for reverse phase and sizeexclusion chromatography.

The medium may also be a membrane comprised of a plurality of aggregatesprovided on a support, which medium is suitable for use in filtration.

In a third aspect, the invention relates to a process for isolating atleast one compound from a sample comprising the steps of contacting saidsample with a separation medium as defined above, or a separation mediumprepared as above, to purify the compound by said medium, for example byadsorption.

Further objects and advantages of the present invention will appear fromthe claims and the detailed description that follows below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of the assembly of latex particles by aninverse suspension process of the invention.

FIG. 2 is a SEM Scanning Electron Microscope photograph of sphericalaggregates of the invention.

DETAILED DESCRIPTION OF INVENTION

The present invention provides methods for designing porous media with afractal structure, starting with small primary particles (nm), which areprogressively clustered into larger aggregates (□m) via a conglomerationprocedure.

The present invention provides a novel approach by using an inversesuspension process (water in oil) from monodisperse latex spheres. Thebasic principles of the method according to the invention areillustrated in FIG. 1.

The first step concerns the synthesis of primary latex particles.Monodisperse latex spheres have been obtained by free-emulsioncopolymerisation between styrene and methacrylic acid, glycidylmethacrylate and aminoethylmethacrylate in order to get functionalisedlatices for further derivatisation. In this way different functionalisedlatices with carboxy, epoxy and amino functions were obtained, varyingthe ratio between comonomers and varying the solids content percentage.

The second step is formation of spherical aggregates by inversesuspension process starting with dispersing the aqueous solution ofprimary latex particles as droplets in oil. Then a fusion betweenfunctionalised latices and an appropriate agent or “glue” should occurin each droplet in order to obtain spherical aggregates with a definedsize. Preferably, fusion is effected by addition of differentwater-soluble compounds acting as a “glue” between particles tostabilise them. Among all the different compounds used as a glue betweenlatex particles, the water-soluble polymer: polyethyleneimine and thecross-linkers N,N′-methylenebisacrylamide and diacryloyl piperazine gavethe best results. This second step makes the aggregates less fragile andeasier to manipulate.

During the aggregation and self-assembly step, thermal fusion of thelatex particles may also be obtained by the high temperature used forthe evaporation.

The inverse suspension process is preferably repeated at least once toform aggregates of latex particles of larger size in the micron range.

The discontinuous phase used is preferably a concentrated latex solution(such as 33% solids content) in order to increase the probability ofaggregate formation. The continuous phase is preferably toluene.

According to the present invention the inverse suspension process iscombined with evaporation to remove water in the aggregates. In apreferred embodiment, the evaporation is carried out from anazeotrope-forming system, so that the mixed water-solvent vapours can becondensed in e.g. a Dean-Stark trap where two separate phases areformed. The solvent phase can then be returned to the suspension, whilethe water phase is removed. This also provides a possibility to monitorthe amount of water removed from the system at a given time.

During the Dean-stark process the temperature is high enough to causethermal fusion of the latex particles and this gives improved mechanicaland solvent stability of the aggregates. The fusion temperature may becontrolled by controlling the comonomer composition.

The third step is size fractionation of aggregates from the second step.This can be done with conventional classification methods, such assieving, sedimentation, elutriation, hydrocyclones etc.

The method of the invention leads to stable and spherical aggregates.The aggregates can be cleaned and dried without destroying the sphericalstructure.

Depending on the starting materials used, the present separation mediumcan be further derivatised with ligands i.e. binding groups suitable forchromatography. Techniques for derivatisation of synthetic polymers,such as latex, are well known in this field, for more details see e.g.a) ton exchangers, Ed K Dorfner, Walter de Gruyter Berlin 1991, b) MLeonard, J Chromatogr B 699, 3-27 (1997), c) Uniform latex particles, LB Bangs, Seradyn Inc, Indianapolis 1984.

A second aspect of the present invention is a separation mediumcomprised of one or more essentially spherical aggregates, wherein eachaggregate comprises a plurality of porous and essentially sphericalpolymer beads assembled into aggregates by an inverse suspensionprocess. The present separation medium can be used e.g. as thestationary phase in chromatography or as a membrane in filtrationprocesses.

In an advantageous embodiment, the polymers have been chemicallycrosslinked. Aggregates comprised of chemically crosslinked polymers mayprovide a more tolerant separation medium than other embodiments, forexample as concerns cleaning and/or packing procedures using roughermethods. In an advantageous embodiment, the pore size and poredistribution within the present medium are similar to the properties ofcommercially available beads of such synthetic polymers, such asstyrene-divinylbenzene beads from Amersham Biosciences, PolymerLaboratories, Perseptive Biosystems etc or methacrylate beads fromTosoh, Merck, Bio-Rad etc.

Accordingly, since the beads that constitute the aggregates are porousin themselves, the aggregates according to the present invention willpresent a hierarchical pore structure.

As regards the size of the aggregates, a minimal number of beads will inpractise be required to form a spherically shaped aggregate, but thisnumber will e.g. depend on the size of the beads. In one embodiment, theaverage aggregate in the present medium is comprised of at least about10 beads in diameter, and preferably at least about 20, such as at least100 beads in diameter. The desired upper limit of the number of beads ineach aggregate will depend on the intended use thereof. Thus, in oneembodiment, the average aggregate in the present medium is comprised ofup to about 400 beads in diameter, such as up to about 200 beads indiameter. Accordingly, the size of the present aggregates can varywithin wide limits, such as from a few μ up to several hundreds or evena thousand μ in particle diameter, e.g. between about 50 and 2000 μm,such as 100-1000 μm.

As mentioned above, the number of beads in each aggregate i.e. the sizeof the aggregate is controlled by the initial size of the primaryparticles as well as on the number of times the inverse suspensionprocess is repeated. Thus, by selection of the appropriate parametersduring the preparation, an aggregate of desired size can be prepared.

In an advantageous embodiment of the present medium, the shape of theaggregates is essentially spherical. A spherical shape is advantageouse.g. in chromatographic processes, since it improves the flow propertiesof an aggregate used as a stationary phase.

The aggregates can comprise one of the above-mentioned kind of beads ora mixture of such beads. Further, the beads can be of essentially thesame size or of sizes that varies within a certain range. Commerciallyavailable beads are generally available either in one defined size or ofa size within a certain range. The intended use of the aggregates willdecide whether beads of one or more sizes are desired.

Thus, the aggregates according to the invention will exhibit equivalentbinding properties as the beads included therein, since the surfaces ofthe beads will also be available in a separation medium comprising suchbeads. Accordingly, a first separation property or function will beprovided by the included beads. Illustrative examples of such propertiesare e.g. ion-exchange ligands, affinity groups, hydrophobicallyinteractive surfaces etc.

In an advantageous embodiment, the present medium has been prepared by amethod as defined above.

As mentioned above, the present medium can e.g. be used inchromatography or in filtration. Thus, the beads may have beenderivatised as discussed above to provide suitable binding groups. As iseasily realised, the nature of the binding groups will decide what kindof chromatography the present separation medium is useful in, e.g. ionexchange chromatography, affinity chromatography, hydrophobicinteraction chromatography etc.

Thus, in one embodiment, the medium is comprised of a plurality of theabove-defined aggregates as separate and essentially spherical entitiesand is suitable for use in packed or expanded bed adsorption. Suchchromatographic methods are well known to the skilled person in thisfield.

In an alternative embodiment, the medium is a membrane comprised of aplurality of said aggregates assembled onto a support and suitable foruse in filtration. The preparation of a membrane from an adsorbingmedium such as the present is well known to the skilled person in thisfield.

A last aspect of the present invention is a process for separating atleast one compound from a liquid comprising the steps of contacting saidliquid with a separation medium according to the invention or preparedaccording the invention to adsorb the compound to said medium. Methodsof chromatography are well-known in this field and the skilled personcan easily adapt the process in suitable ways. In brief, in a firststep, a solution comprising a desired compound is passed over aseparation medium according to the invention under conditions allowingadsorption of the compound to ligands i.e. binding groups present onsaid matrix. Such conditions are controlled e.g. by pH and/or saltconcentration i.e. ionic strength in the solution. Care should be takennot to exceed the capacity of the medium, i.e. the flow should besufficiently slow to allow a satisfactory adsorption. In this step,other components of the solution will pass through in principleunimpeded. Optionally, the medium is then washed, e.g. with an aqueoussolution, in order to remove retained and/or loosely bound substances.In a next step, a second solution denoted an eluent is passed over themedium under conditions that provide desorption i.e. release of thedesired compound. Such conditions are commonly a decrease of the pHand/or an increase of the salt concentration i.e. ionic strength. As thepH drops, the net charge on the compound will change as it becomes morepositive, and hence alter many of the opportunities that it has forelectrostatic interactions. Similarly, the increase of the ionicstrength by addition of a salt will also alter the affinity between thecompound and the ligand. If more than one compounds are present in theliquid, other compounds than the desired one may adsorb to the medium.The desired compound and any further compound(s) will subsequently beavailable for selective elution since they desorb from the medium atdifferent conditions.

The desired compound may be a any compound, such as a recombinantlyproduced protein, peptide, nucleic acid, virus etc, or alternatively anundesired contaminant, such as an organic compound, which it is desiredto remove from a liquid.

As an illustrative example of separation of a desired target compound,plasmid purification is mentioned. In this case, the relatively largesize of the pores of the aggregates of the present separation medium isadvantageous, such as 0.5-2 μm.

Another application where the present invention may prove especiallyadvantageous is in expanded bed adsorption chromatography, where acertain minimum density of the particles is desired. Such a density iseasily provided by the aggregates according to the invention.

Additional applications of the present aggregates are e.g. as carriersin cell culture, in which case larger beads are advantageous, optionallyof a relatively wide spread size distribution, or as carriers in variousdelivery systems, such as drug delivery.

EXAMPLES

The present examples are provided for illustrative purposes only andillustrate certain embodiments of the instant invention. They are notintended to be illustrative of all embodiments of the present inventionas recited in the claims.

I. Synthesis of Primary Latex Particles

General Procedure for Emulsion Polymerisation

Distilled and deionised water is added in a four-necked double jacketreactor (250 mL) filled with a nitrogen bubbler, stirring paddle andcondenser. Nitrogen is bubbled through the water until the system reachthermal equilibrium at the reaction temperature. Monomers are thenintroduced and the mixture is vigorously stirred and purged withnitrogen for 15 min (430 rpm). The initiator is then added, dissolved ina known amount of water. The flow of nitrogen to the bubbler was thenreduced to minimize the stripping of the monomer from the reactionmixture and the stirring rate is reduced (350 rpm). The mixture isstirred and allowed to react for 24 h at constant temperature. After 24h, the reaction mixture is cooled and filtered through glass wool toremove any coagulum.

Carboxy-Functional Latex: Poly(Styrene/Methacrylic Acid),

Reaction temperature=70° C.,

Initiator=ammonium persulfate, C=1.4 10⁻³ mol.L⁻¹

Epoxy-Functional Latex: Poly(Styrene/Glycidyl Methacrylate),

Reaction temperature=65° C.

Initiator=potassium persulfate, C=2.07 10⁻³ mol.L³¹ ¹

Amino-Functional Latex: Poly(Styrene/AminoethylmethacrylateHydrochloride)

Reaction temperature=70° C.

Initiator=V50 (2,2′-azobis(2-amidinopropane)dihydrochloride, C=5.89 10⁻³mol.L⁻¹

Chemical Modification of Carboxy-Functional Latex (Styrene:MA: 90:10)

After centrifugation twice in distilled water of 1.5 mL of latex, thiswas diluted in 6 mL of distilled water and 1 equivalent of amino ethylmethacrylate hydrochloride (AEMH) (m=0.024 g) and 1 equivalent of EDC(m=0.0367 g) were added. Then, pH was adjusted to 6-7 with hydrochloricacid 1 M. Then the mixture is stirred at 4° C. during 2 hours. Then, themixture is let at room temperature overnight. After the end of thereaction, the mixture is centrifuged several times with 0.1 M NaClsolution.

This procedure provides a crosslinkable shell and a hard core of theprimary particle.

II. Fusion of Primary Particles and Formation of Spherical Aggregates byInverse Suspension Polymerisation

Inverse suspension polymerisation is a copolymerisation processconducted in a dispersed aqueous phase containing all the ingredientsneeded for the formation of the network. The resulting sphericalparticles are easily removed by filtration or centrifugation from thecontinuous organic phase. The discontinuous phase used is a concentratedlatex solution (33% solids content) in order to increase the probabilityof aggregate formation.

General Procedure for Large Scale Inverse Suspension Process withDean-Stark Distillation Apparatus

Latex (4.5 mL: 1.485 g) is centrifuged twice in distilled water. Forsome experiments, defined amounts of water-soluble compounds and ofinitiator have been added to the aqueous solution of latex. Thediscontinuous phase obtained is let at room temperature. Continuousphase is prepared by dissolving sorbitan monooleate (2 g, C=0.1037 M) in45 mL of toluene. Then this mixture is purged with nitrogen untiltemperature reached its equilibrium (T=70° C.). Agitation is maintainedat 500 rpm. Then the discontinuous phase is added. After the formationof the aggregates after half an hour, we start to increase thetemperature until Dean-Start distillation temperature (T=105° C.).During the aggregation and self-assembly step, thermal fusion of thelatex particles occurs. After the reaction had been cooled, thesupernatant is removed and then some clean toluene is added. Theaggregates are then cleaned with some heptane and dried in an oven at40° C.

General Procedure for the Scale-Up of the Inverse Suspension Processwith Dean-Stark Distillation Apparatus

Latex (30 mL: 9.9 g) is centrifuged twice in distilled water. A definedamount of N,N′-methylenebisacrylamide and of the initiator ammoniumpersulfate have been added to the aqueous solution of latex. Thediscontinuous phase obtained is let at room temperature. Continuousphase is prepared by dissolving sorbitan monooleate (13.33 g, C=0.1037M) in 300 mL of toluene. Then this mixture is purged with nitrogen untiltemperature reached its equilibrium (T=70° C.). Agitation is maintainedat 500 rpm. Then the discontinuous phase is added. After the formationof the aggregates after half an hour, we start to increase thetemperature until Dean-Start distillation temperature (T=115° C.). Afterthe reaction had been cooled, the supernatant is removed and then someclean toluene is added. The aggregates are then cleaned with someheptane and dried in an oven at 40° C. The aggregates (see FIG. 2) areseparated by sizes using sieves.

The method of the invention is repeated a desired number of times withmonosized aggregates, depending on the desired aggregate size of thefinished product. In the last round of aggregation the aggregates do nothave to be sieved but can be used without the last sieving step ifdesired.

Thus, the invention provides a fully controllable method of achievinghierarchical structures. The invention provides full control of the sizeat any level. Furthermore, the spherical aggregates are fully permeableand accessible for chromatography purposes.

Those skilled in the art, having the benefit of the teachings of thepresent invention as set forth above, can effect numerous modificationsthereto. These modifications are to be construed as being encompassedwithin the scope of the present invention as set forth in the appendedclaims.

1. A method of preparing a separation medium starting with an aqueoussolution of essentially spherical primary particles of a defined size,comprising the following steps: a) inverse suspension dispersing intooil said aqueous solution of primary particles to form a droplet in oildispersion; b) evaporating to remove water in said aqueous solutiondispersion and to encourage fusion between particles to form sphericalaggregates; c) size fractionating of aggregates from step b); andoptionally d) suspending the aggregates from step c) in aqueous solutionand repeating steps a) to c) an optional number of times to formprogressively larger spherical aggregates.
 2. The method of claim 1,wherein the primary particles comprise a synthetic polymer selected fromthe group consisting of polymerised styrene and/or divinylbenzene,(meth)acrylates, vinyl esters, vinyl ethers, vinyl amides,meth(acrylamides), dienes and mixtures of two or more of the foregoing.3. The method of claim 1, wherein the primary particles are 50-1000 nmin diameter.
 4. The method of claim 1, wherein the primary particles arefunctionalised.
 5. The method of claim 4, wherein the fusion in step b)is between functional groups on the primary particles and the fusion iscaused by a water-soluble polymer and a cross-linker.
 6. The method ofclaim 1, wherein the fusion in step b) is by thermal fusion of theparticles.
 7. The method of claim 4, wherein the water-soluble polymeris polyethyleneimine and the cross-linker is chosen fromN,N′-methylenebisacrylamide and diacryloyl piperazine.
 8. The method ofclaim 1, wherein the evaporation in step b) is a Dean-Starkdistillation.
 9. The method of claim 1, further comprising, in a stepbefore step a), derivatizing functional groups of the polymers gel toprovide a separation medium, which exhibits one or two different kind ofligands.
 10. A separation medium comprised of one or more aggregates,wherein each aggregate comprises a plurality of porous and essentiallyspherical synthetic polymer beads assembled into essentially sphericalaggregates of controlled size.
 11. The medium of claim 10, wherein themass ratio of synthetic polymer:beads in each aggregate is about 0.1:100to 10:100.
 12. The medium of claim 10, wherein the beads comprises asynthetic polymer selected from the group consisting of polymerisedstyrene and/or divinylbenzene, (meth)acrylates, vinyl esters, vinylethers, vinyl amides, meth(acrylamides), dienes and a mixture of two ormore of the foregoing.
 13. The medium of claim 10, wherein the polymershave been chemically cross-linked.
 14. The medium of claim 10, whereinthe polymers have been thermally fused.
 15. The medium of claim 10,which exhibits one kind or two different kinds of ligands coupled to thebeads within the aggregates.
 16. The medium of claim 10, wherein eachaggregate comprises at least about 10 to up to 400 beads.
 17. The mediumof claim 10, wherein the aggregates are separate and essentiallyspherical entities.
 18. The medium of claim 10, comprising a membraneincluding a plurality of aggregates provided on a support.